Spi1 aggravates neuropathic pain by modulating Clec7a-mediated neuroinflammation and microglial phagocytosis.

Background: Microglia are essential in mediating responses to nerve injury, with neuroinflammation and engulfment of interneuron inhibitory synapses driving heightened neuronal excitability and pain hypersensitivity. Spi1 is the main regulator of microglial homeostasis in the central nervous system. However, its function in neuropathic pain remains unclear. The aim of this study was to investigate the effect of Spi1 on neuropathic pain and the underlying mechanism.

Methods: RNA-seq analysis identified Spi1 as a central hub gene in neuropathic pain. The spared nerve injury (SNI) mice and LPS induced BV2 cells were employed to detect the expression of Spi1. Silencing Spi1 was performed to determine their roles in SNI progression. Mechanical allodynia and thermal allodynia were assessed using von Frey filaments and hot plate, respectively. The expression of pro-inflammation marker CD86, anti-inflammation marker CD206 and inflammatory cytokines (TNF-α, IL-1β, IL-6) were qualified by western blot, immunofluorescence or qPCR. Additionally, the microglia morphological changes, the volume of CD68 or VGAT in microglia were examined by immunofluorescence to reflect microglial activation and phagocytosis. Patch clamp was performed to evaluate neuronal excitability. Luciferase assays and ChIP-qPCR were conducted to pinpoint the binding between Spi1 and the promoter of Clec7a. Subsequently, rescue experiments in vivo and in vitro were implemented to clarify whether Spi1 exerted itself through Clec7a.

Results: Elevated Spi1 expression was observed in the spinal cords of SNI mice and in LPS-stimulated BV2 cells. Spi1 knockdown alleviated SNI-induced mechanical and thermal allodynia, diminished the release of inflammatory cytokine and the expression of proinflammation marker CD86, decreased the volume of CD68 or VGAT in microglia and increased the frequency of neuronal spontaneous inhibitory postsynaptic potentials(sIPSCs). Mechanistically, Spi1 was found to bind the promoter of Clec7a by luciferase assays and ChIP-PCR. And rescue experiments in vivo and in vitro showed that Spi1 aggravated pain hypersensitivity, promoted neuroinflammation and enhanced microglial phagocytosis by Clec7a.

Conclusion: Our results highlight that Spi1-Clec7a axis was a key contributor to neuropathic pain through promoting neuroinflammation and microglial phagocytosis, thereby implicating it as a promising therapeutic target for mitigating neuropathic pain.

Supplementary Information: The online version contains supplementary material available at 10.1186/s10194-025-02089-x.